Assays for dissecting the in vitro enzymatic activity of yeast Ubc7

Bayan Mashahreh, Yuval Reiss, Reuven Wiener, Tommer Ravid*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Scopus citations

Abstract

Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or endoplasmic reticulum-associated degradation (ERAD). Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10, and the Asi complex, which resides at the nuclear envelope (NE). These ER/NE membrane-associated RING-type E3 ligases recognize and ubiquitylate defective proteins in cooperation with the E2 conjugating enzyme Ubc7 and the obligatory Ubc7 cofactor Cue1. Interaction of Ubc7 with the RING domains of its cognate E3 Ub-protein ligases stimulates the formation of isopeptide (amide) Ub–Ub linkages. Each isopeptide bond is formed by transfer of an Ubc7-linked activated Ub to a lysine side chain of an acceptor Ub. Multiple Ub transfer reactions form a poly-Ub chain that targets the conjugated protein for degradation by the proteasome. To study the mechanism of Ub–Ub bond formation, this reaction is reconstituted in a cell-free system consisting of recombinant E1, Ub, Ubc7, its cofactor Cue1, and the RING domain of either Doa10 or Hrd1. Here we provide detailed protocols for the purification of the required recombinant proteins and for the reactions that produce an Ub–Ub bond, specifically, the formation of an Ubc7 ~ Ub thiolester (Ub charging) and subsequent formation of the isopeptide Ub–Ub linkage (Ub transfer). These protocols also provide a useful guideline for similar in vitro ubiquitylation reactions intended to explore the mechanism of other Ub-conjugation systems.

Original languageAmerican English
Title of host publicationUbiquitin-dependent Protein Degradation
EditorsMark Hochstrasser
PublisherAcademic Press Inc.
Pages71-95
Number of pages25
ISBN (Print)9780128186671
DOIs
StatePublished - 1 Jan 2019

Publication series

NameMethods in Enzymology
Volume619
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
We wish to thank Dr. A. Shiber and Dr. I Cohen for their immense contribution to setting up the protein purification and the in vitro assays. The authors thank A. Ciechanover, R. Hampton, and M. Hochstrasser for plasmids. This work is kindly supported by the Israel Science Foundation (grants 786/08, 119/14).

Publisher Copyright:
© 2019 Elsevier Inc.

Keywords

  • E1 ubiquitin-activating enzyme
  • E2 ubiquitin-conjugating enzyme
  • E3 ubiquitin ligase
  • In vitro ubiquitylation
  • Recombinant proteins
  • The Doa10 ubiquitylation complex
  • Ubiquitin

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